Limits...
Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.

Santiago-Martínez E, Soplop NH, Patel R, Kramer SG - J. Cell Biol. (2008)

Bottom Line: Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects.In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane.In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.

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LOF and GOF phenotypes in the heart. All embryos are stage 17. (A) Dorsal view of a wild-type embryo stained for Mef2 (magenta) and Slit (green). (B) EM of a wild-type heart in XS showing the lumen between two CBs (arrow). (C) robo embryo stained for Slit (green) and Mef2 (magenta). (D) EM of a robo embryo showing a block in lumen formation. Arrow indicates the region where the CBs remain inappropriately attached. (E) XS of a slit GOF embryo stained with anti-Mef2, which labels CB nuclei. Two lumens are visible (arrowheads). (E′) Removal of robo in a slit GOF background suppresses the two-lumen phenotype. (F) Anti–E-Cad staining in the wild-type heart. Staining at the ventral attachment point is marked with an arrow. (G) EM of shg/E-Cad mutant shows that CBs fail to attach to each other (bracket). (H and H′) Close up of EMs in G and D. In robo embryos (H′), the CBs are closely associated (arrow), as compared with shg/E-Cad mutants (H), where the cells fail to adhere, as indicated by the presence of extracellular space between CBs (arrow). (I) EM of a shg/+,robo/+ embryo showing defects in lumen formation. (J) EM of a Mef-Gal4/+;UAS-shg/+ embryo showing that lumen formation is blocked (arrow). (K) Mef-Gal4/+;UAS-robo/+ embryo stained for Slit (green) and Mef2 (magenta). Slit is localized to the apical domain of CBs but the staining is nonuniform (arrows). (L) EM of a Mef-Gal4/+;UAS-robo/+ embryo in which the CBs have lost their dorsal point of attachment (arrows). Bars: (B) 2 μm; (H) 1 μm.
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fig3: LOF and GOF phenotypes in the heart. All embryos are stage 17. (A) Dorsal view of a wild-type embryo stained for Mef2 (magenta) and Slit (green). (B) EM of a wild-type heart in XS showing the lumen between two CBs (arrow). (C) robo embryo stained for Slit (green) and Mef2 (magenta). (D) EM of a robo embryo showing a block in lumen formation. Arrow indicates the region where the CBs remain inappropriately attached. (E) XS of a slit GOF embryo stained with anti-Mef2, which labels CB nuclei. Two lumens are visible (arrowheads). (E′) Removal of robo in a slit GOF background suppresses the two-lumen phenotype. (F) Anti–E-Cad staining in the wild-type heart. Staining at the ventral attachment point is marked with an arrow. (G) EM of shg/E-Cad mutant shows that CBs fail to attach to each other (bracket). (H and H′) Close up of EMs in G and D. In robo embryos (H′), the CBs are closely associated (arrow), as compared with shg/E-Cad mutants (H), where the cells fail to adhere, as indicated by the presence of extracellular space between CBs (arrow). (I) EM of a shg/+,robo/+ embryo showing defects in lumen formation. (J) EM of a Mef-Gal4/+;UAS-shg/+ embryo showing that lumen formation is blocked (arrow). (K) Mef-Gal4/+;UAS-robo/+ embryo stained for Slit (green) and Mef2 (magenta). Slit is localized to the apical domain of CBs but the staining is nonuniform (arrows). (L) EM of a Mef-Gal4/+;UAS-robo/+ embryo in which the CBs have lost their dorsal point of attachment (arrows). Bars: (B) 2 μm; (H) 1 μm.

Mentions: In embryos mutant for slit or both robo and robo2, the rows of CBs do not properly align, resulting in gaps at the dorsal midline (Santiago-Martínez et al., 2006). It has been found that slit mutant embryos have lumen defects (MacMullin and Jacobs, 2006). However, because of the severe CB alignment phenotype in slit or robo,robo2 mutants, we found it difficult to separate slit's earlier role in ipsilateral CB cell alignment with what we believe to be a later role lumen formation between contralateral CBs. Embryos mutant for robo alone have very mild migration defects, and the majority of CBs are able to align at the dorsal midline (Santiago-Martínez et al., 2006). EM of robo mutants (n = 11) reveals that contralateral CBs are inappropriately adhered, and the lumen fails to form between these cells (Fig. 3 D). In addition, Slit is no longer concentrated at CB apical domains (Fig. 3 C), which suggests that the Robo localization is important for the polarized accumulation of Slit. Moreover, the mislocalization of Slit in a robo mutant is not sufficient to induce ectopic lumen formation as in slit GOF embryos (Fig. 2 F), which suggests that Robo is required for this process. To further explore the functional connection between Slit and Robo, we show that removal of robo in slit GOF embryos completely suppresses the slit GOF phenotype and blocks lumen formation (Fig. 3, E and E′). Finally, we found that Dlg and α-spectrin are properly localized in robo mutants (Fig. S1, E and J), providing additional evidence that during lumen formation, Slit and Robo are not required for CB cell polarity but instead function at a later step once the initial polarity of the cell has already been established.


Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.

Santiago-Martínez E, Soplop NH, Patel R, Kramer SG - J. Cell Biol. (2008)

LOF and GOF phenotypes in the heart. All embryos are stage 17. (A) Dorsal view of a wild-type embryo stained for Mef2 (magenta) and Slit (green). (B) EM of a wild-type heart in XS showing the lumen between two CBs (arrow). (C) robo embryo stained for Slit (green) and Mef2 (magenta). (D) EM of a robo embryo showing a block in lumen formation. Arrow indicates the region where the CBs remain inappropriately attached. (E) XS of a slit GOF embryo stained with anti-Mef2, which labels CB nuclei. Two lumens are visible (arrowheads). (E′) Removal of robo in a slit GOF background suppresses the two-lumen phenotype. (F) Anti–E-Cad staining in the wild-type heart. Staining at the ventral attachment point is marked with an arrow. (G) EM of shg/E-Cad mutant shows that CBs fail to attach to each other (bracket). (H and H′) Close up of EMs in G and D. In robo embryos (H′), the CBs are closely associated (arrow), as compared with shg/E-Cad mutants (H), where the cells fail to adhere, as indicated by the presence of extracellular space between CBs (arrow). (I) EM of a shg/+,robo/+ embryo showing defects in lumen formation. (J) EM of a Mef-Gal4/+;UAS-shg/+ embryo showing that lumen formation is blocked (arrow). (K) Mef-Gal4/+;UAS-robo/+ embryo stained for Slit (green) and Mef2 (magenta). Slit is localized to the apical domain of CBs but the staining is nonuniform (arrows). (L) EM of a Mef-Gal4/+;UAS-robo/+ embryo in which the CBs have lost their dorsal point of attachment (arrows). Bars: (B) 2 μm; (H) 1 μm.
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Related In: Results  -  Collection

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Show All Figures
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fig3: LOF and GOF phenotypes in the heart. All embryos are stage 17. (A) Dorsal view of a wild-type embryo stained for Mef2 (magenta) and Slit (green). (B) EM of a wild-type heart in XS showing the lumen between two CBs (arrow). (C) robo embryo stained for Slit (green) and Mef2 (magenta). (D) EM of a robo embryo showing a block in lumen formation. Arrow indicates the region where the CBs remain inappropriately attached. (E) XS of a slit GOF embryo stained with anti-Mef2, which labels CB nuclei. Two lumens are visible (arrowheads). (E′) Removal of robo in a slit GOF background suppresses the two-lumen phenotype. (F) Anti–E-Cad staining in the wild-type heart. Staining at the ventral attachment point is marked with an arrow. (G) EM of shg/E-Cad mutant shows that CBs fail to attach to each other (bracket). (H and H′) Close up of EMs in G and D. In robo embryos (H′), the CBs are closely associated (arrow), as compared with shg/E-Cad mutants (H), where the cells fail to adhere, as indicated by the presence of extracellular space between CBs (arrow). (I) EM of a shg/+,robo/+ embryo showing defects in lumen formation. (J) EM of a Mef-Gal4/+;UAS-shg/+ embryo showing that lumen formation is blocked (arrow). (K) Mef-Gal4/+;UAS-robo/+ embryo stained for Slit (green) and Mef2 (magenta). Slit is localized to the apical domain of CBs but the staining is nonuniform (arrows). (L) EM of a Mef-Gal4/+;UAS-robo/+ embryo in which the CBs have lost their dorsal point of attachment (arrows). Bars: (B) 2 μm; (H) 1 μm.
Mentions: In embryos mutant for slit or both robo and robo2, the rows of CBs do not properly align, resulting in gaps at the dorsal midline (Santiago-Martínez et al., 2006). It has been found that slit mutant embryos have lumen defects (MacMullin and Jacobs, 2006). However, because of the severe CB alignment phenotype in slit or robo,robo2 mutants, we found it difficult to separate slit's earlier role in ipsilateral CB cell alignment with what we believe to be a later role lumen formation between contralateral CBs. Embryos mutant for robo alone have very mild migration defects, and the majority of CBs are able to align at the dorsal midline (Santiago-Martínez et al., 2006). EM of robo mutants (n = 11) reveals that contralateral CBs are inappropriately adhered, and the lumen fails to form between these cells (Fig. 3 D). In addition, Slit is no longer concentrated at CB apical domains (Fig. 3 C), which suggests that the Robo localization is important for the polarized accumulation of Slit. Moreover, the mislocalization of Slit in a robo mutant is not sufficient to induce ectopic lumen formation as in slit GOF embryos (Fig. 2 F), which suggests that Robo is required for this process. To further explore the functional connection between Slit and Robo, we show that removal of robo in slit GOF embryos completely suppresses the slit GOF phenotype and blocks lumen formation (Fig. 3, E and E′). Finally, we found that Dlg and α-spectrin are properly localized in robo mutants (Fig. S1, E and J), providing additional evidence that during lumen formation, Slit and Robo are not required for CB cell polarity but instead function at a later step once the initial polarity of the cell has already been established.

Bottom Line: Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects.In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane.In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.

Show MeSH
Related in: MedlinePlus